Molecular Characterization of Klebsiella pneumoniae Clinical Isolates with Elevated Resistance to Carbapenems

BACKGROUND: Emergence of carbapenems-resistant K. pneumoniae represents a serious challenge for antimicrobial therapy. OBJECTIVE: The aim of this research is to determine different mechanisms mediating the emergence of K. pneumoniae isolates with high-level carbapenem resistance. METHOD: A total of 80 K. pneumoniae isolates were purified from sputum and urine specimens. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by broth microdilution method. Carbapenemases were detected by Modified Hodge test and PCR. Additionally, the copy numbers of the identified genes (bla(VIM-1), bla(NDM-1) and bla(OXA-48)) were quantified by RT-PCR. The outer membrane proteins OmpK35 and OmpK36 of the resistant isolates were analyzed. RESULTS: Eight isolates were resistant to carbapenems; six of these isolates possessed elevated MICs to imipenem and meropenem (≥16 µg/ml). Carbapenem resistant isolates harbored bla(NDM-1) (n=5), bla(VIM-1) (n=4) and bla(OXA-48) (n=1) with some isolates had multiple carbapenemases genes. Six isolates with high MICs to imipenem contained multi-copies of the carbapenemases genes along with the lack of OmpK35. Isolates with intermediate resistance to carbapenems (MIC; 4-8 µg/ml) did not exhibit multiple carbapenemases but lacked the OmpK35. Random amplified polymorphic DNA exhibited three different patterns and indicated that five isolates encoded the same pattern P1. CONCLUSION: This study elucidated that multiple carbapenemases genes, high copy number of carbapenemases and loss of the porin OmpK35 could collectively contribute to the emergence of K. pneumoniae isolates with high resistance to carbapenems. Hence, more restrictions should be applied on the use of carbapenems to reduce the emergence of the resistant clones.